Colloidal gold is a dispersion of gold nanoparticles in a dispersion medium, typically water but other medium can also be used as discussed below. Gold nanoparticles have attracted substantial interest from scientists for over a century because of their unique physical, chemical, and surface properties, such as: (i) size- and shape-dependent strong optical extinction and scattering which is tunable from ultraviolate (UV) wavelengths all the way to near infrared (NIR) wavelengths; (ii) large surface areas for conjugation to functional ligands; and (iii) little or no long-term toxicity or other adverse effects in vivo allowing their high acceptance level in living systems. Colloidal gold nanoparticles, also referred to in the present specification and claims as gold nanocolloids, are now being widely investigated for its potential use in a wide variety of biological and medical applications. Applications include use as an imaging agent, a sensing agent, a gene-regulating agent, a targeted drug delivery carrier, and in photoresponsive therapeutics. Most of these applications require the colloidal gold undergo surface modification, also referred to as surface functionalization in the specification and claims of the present invention, prior to its use in the application.
Currently, the overwhelming majority of gold nanocolloids are prepared by using the standard wet chemical sodium citrate reduction of tetrachloroaurate (HAuCl4) methodology. This method results in the synthesis of spherical gold nanoparticles with diameters ranging from 5 to 200 nm which are capped or covered with negatively charged citrate ions. The citrate ion capping prevents the nanoparticles from aggregating by providing electrostatic repulsion. Once formed and prior to their use in biological and medical applications the sodium citrate capped gold nanoparticles must undergo further surface functionalization, usually via conjugation of functional ligand molecules to the surface of the nanoparticle.
Other wet chemical methods for formation of colloidal gold include the Brust method, the Perrault method and the Martin method. The Brust method relies on reaction of chlorauric acid with tetraoctylammonium bromide in toluene and sodium borohydride. The Perrault method uses hydroquinone to reduce the HAuCl4 in a solution containing gold nanoparticle seeds. The Martin method uses reduction of HAuCl4 in water by NaBH4 wherein the stabilizing agents HCl and NaOH are present in a precise ratio. All of the wet chemical methods rely on first converting gold (Au) with strong acid into the atomic formula HAuCl4 and then using this atomic form to build up the nanoparticles in a bottom-up type of process. All of the methods require the presence of stabilizing agents to prevent the gold nanoparticles from aggregating and precipitating out of solution. Once the stabilized nanoparticles are formed further surface functionalization must occur before the nanoparticles can be used in their many potential applications. These surface modifications also must not result in destabilization of the colloidal suspension and precipitation of the gold nanoparticles. Ligand exchange reactions have been found to be a particularly powerful approach for surface modification of various inorganic colloidal nanoparticles including gold nanocolloids and they are widely used to produce organic and water-soluble nanoparticles with various core materials and functional groups.
One of the most difficult aspects of carrying out ligand exchange reactions involving colloidal gold nanoparticles is maintaining the stability of the colloidal suspension of gold during the reaction. As reflected in a large number of reported protocols, Woehrle, G. H., Brown, L. O., and Hutchison, J. E., J. Am. Chem. Soc., Vol. 127 (2005), 2172-2183; Liao, H., and Hafner J. H., Chem. Mater., Vol. 17 (2005), 4636-4641; Ojea-Jimenez, I. and Puntes, V., J. Am. Chem. Soc., Vol. 131 (2009), 13320-13327, to ensure completion of the ligand exchange reaction and to avoid precipitation of gold nanoparticles it is often necessary to use a very large excess, sometimes over a 10 fold excess, of the ligand over the amount required to form a monolayer, as found by referring to the literature values for the ligand footprint on gold surfaces, on the surface of the nanoparticles. It is undesirable to have the excess unreacted free ligand left in the gold nanocolloids since it might interfere with or alter the expected functionalities of the gold nanoparticle conjugates formed. It is not easy, however, to remove the excess free ligand without inducing aggregation or leading to a noticeable loss of gold nanoparticle conjugates. It is also very difficult to create gold nanoparticles with more than one type of ligand bound to them when starting from wet chemical produced colloidal gold. Because the ligands must be added in such a large excess, generally a 1000% excess over the amount required to form a monolayer based on footprint analysis, it is not possible with current colloidal gold systems to prepare gold nanoparticles either with a defined number of ligands per nanoparticle, which would be very beneficial for many applications and fundamental studies, or with multiple ligands. Finally, because of the low conjugation efficiency, the ligand exchange reaction is not a good method for the conjugation of precious biomolecules, such as aptamers or vectors, onto gold nanoparticles.
In the present invention, we provide a top-down fabrication method to address the issues described above. In the present specification and claims a top-down fabrication method means a method which begins with a bulk material, not an atomic form of gold as in wet chemical methods, in a liquid and fabricates a stable colloidal nanoparticle suspension in the liquid. In a preferred top-down method of the present invention the method starts with a bulk gold material in a liquid and produces pure, bare, stable gold nanoparticles in a colloidal gold suspension. The produced nanoparticles are bare because the method does not require any stabilizers nor does it involve any citrate reduction. The produced gold nanoparticles can undergo surface modification and the amount of surface coverage by modifying ligands can be tuned to be any percent value between 0 and 100%. The method also allows us to fabricate gold nanoparticles conjugated with multiple types of ligands with different functions, for example, sensing, imaging, improving solubility, and preventing non-specific binding. The method does not require a large excess amount of ligand to prevent aggregation of the gold nanoparticles, thus it is adaptable to use with ligands that are in short supply or very expensive. Because the nanoparticles are bare the conjugation reaction does not involve any competition with or displacement of stabilizers such as citrate as in prior art methods. Thus, even ligands with low affinity for the gold nanoparticles can be used in the conjugation reaction.